Parkinson's disease (PD) is characterized by progressive and selective loss of dopaminergic neurons in substantia nigra pars compacta. Although the pathogenesis of PD remains unclear, a breakthrough on this question has emerged from studies using animal models induced by Parkinsonian neurotoxins, such as MPTP, or viral-targeted overexpression of the PD-related brain-specific protein 1-Synuclein in the SNc. Evidence from different models suggests that the degeneration of dopaminergic neurons in PD is mediated at least in part via apoptosis-execution cascades. In addition, the neurotoxic activation of microglia may also contribute to dopaminergic neurodegeneration in PD. Thus, the long-term goal of this proposal is to identify novel neuroprotective agents that are capable of blocking the apoptosis-signaling pathways in dopaminergic neurons and the neurotoxic activation of microglia induced by Parkinsonian neurotoxins. HSP27 is a member of the small heat shock protein family, a group of ubiquitous stress proteins that are expressed in virtually all organisms. The expression of HSP27 is markedly induced in the brain after several forms of injury, and its neuroprotective role has been demonstrated. In addition to its known function as a protein chaperone, HSP27 has potent anti-apoptotic effects. It appears that HSP27 gains its anti-apoptotic properties after undergoing serine-specific phosphorylation, which can be induced by oxidative stress. However, the precise mechanism underlying the anti-apoptotic effect of HSP27 is not fully understood. We have created transgenic mice overexpressing either the wild-type HSP27 or a non-phosphorylatable HSP27 mutant. Using both transgenic and gene-transfection approaches, we have obtained exciting preliminary results which suggest that: 1) overexpression of HSP27 protects against dopaminergic cell death induced by Parkinsonian neurotoxins or by targeted overexpression of the neurotoxic human mutant (A53T) 1- Synuclein; 2) the neuroprotective effect of HSP27 is dependent on phosphorylation-mediated activation of the protein; and 3) HSP27 may achieve its neuroprotective effects via inhibiting both the ASK1/JNK-dependent mitochondrial death-signaling pathway in dopaminergic neurons and ASK1/p38-dependent pro-inflammatory reactions of microglia. This proposal attempts to further explore HSP27 as a neuroprotective molecule against dopaminergic cell death, with future therapeutic implications. The overall hypothesis underlying this proposal is that enhanced expression and phosphorylation- dependent activation of HSP27 protects against dopaminergic cell death via novel anti-apoptotic and anti-inflammatory mechanisms. We propose to test the hypothesis using both in vivo and in vitro models of PD. While the in vivo animal models mimic some important aspects of pathophysiological changes in PD, the in vitro cellular models will complement the in vivo studies by allowing for precise mechanistic studies. The following specific objectives are proposed: Aim 1. Test the hypothesis that transgenic overexpression and phosphorylation-dependent activation of HSP27 protects against dopaminergic cell death in the brain following Parkinsonian insults. Aim 2. Test the hypothesis that the direct neuroprotective effect of HSP27 against dopaminergic cell death is mediated via a novel anti-apoptotic mechanism involving the disruption of ASK1/JNK-dependent mitochondrial signaling pathways. Aim 3. Test the hypothesis that HSP27 protects against dopaminergic cell death via a second mechanism which involves the disruption of ASK1/p38- dependent pro-inflammatory signaling in brain microglia.